Project Summary The ?brain-eating amoeba? Naegleria fowleri is an NIAID Priority Category B Pathogen that carries a 95% fatality rate, yet the mechanisms underlying its basic biology and pathogenic behaviors remain largely unstudied. Because understanding the cell biology of Naegleria is critical to the development of therapeutics, my long-term goal is to define the cellular and molecular basis of Naegleria pathogenesis. Unlike human cells, from which Naegleria diverged 1-2 billion years ago, these amoebae do not possess cytoplasmic microtubules. This suggests that the actin cytoskeleton, assembled by the Arp2/3 complex and formin family proteins, is the primary driving force for many cellular processes essential to cell survival and pathogenesis. Therefore, my overall objective in this application is to determine how actin cytoskeletal rearrangements promote cell motility, which is important for establishing infection, contractile vacuole pumping, which is required for surviving osmotic pressure, and cell division, which is critical for robust colonization in the brain. To achieve this objective, I propose the following specific aims: (Aim 1) determine the Arp2/3 complex activators driving cell motility, (Aim 2) define the formin-based mechanisms governing contractile vacuole dynamics, and (Aim 3) identify the actin nucleators and molecular mechanisms responsible for cytokinesis. I will address these aims using orthogonal cellular perturbations (small molecule inhibitors to impair the Arp2/3 complex and formin family proteins, as well as individual gene targeting using morpholinos) and environmental perturbations by changing external osmotic pressure. I will measure phenotypes of single cells in detail with microscopy, and complement this by collecting quantitative data on cell populations with flow cytometry and gene expression assays. Because the cell biology underlying pathogenesis and basic functions in Naegleria is severely understudied, defining how actin orchestrates motility, division, and contractile vacuole dynamics is critical for uncovering drug targets to treat these deadly infections. Completing this project will not only advance the mission of the NIH, but it will also prepare me for a career as an independent investigator at a research-intensive university studying the cell biology of Naegleria. The career development resources available to postdocs and the excellent core facility trainings at UMass create an ideal institutional environment for my training. Further, the mentorship of Dr. Fritz- Laylin, an expert on Naegleria biology, and Dr. Wadsworth, who specializes in cell division, will ensure the success of this project as well as my transition to a career as an independent researcher.